The invention relates to amino acid transporter molecules useful in the diagnosis and treatment of disorders of the central and peripheral nervous systems.
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Synaptic transmission in the central and peripheral nervous systems involves the regulated release from presynaptic terminals (exocytosis) of vesicles filled with neurotransmitter. Classical adrenergic and cholinergic neurotransmitters are synthesized in the cytoplasm and transported into the vesicles by active transport mechanisms in which intravesicular H+ ions are exchanged for cytoplasmic transmitter.
Vesicular transport proteins for acetylcholine (ACh) and monamines have been characterized and are known in the art. Transport of these molecules depends primarily on the chemical component (xcex94pH) of the electrochemical gradient, xcex94FH+. Molecular cloning has also demonstrated that the vesicular monoamine and ACh transporters are closely related in structure (Liu, 1992b; Erickson, et al., 1993; Alfonso, et al., 1993).
In contrast, it is now recognized that vesicular transport of the amino acid neurotransmitter glutamate depends primarily on the electrical component (xcex94xcexa8) of xcex94FH+, while vesicular GABA transport appears to depend more equally on both xcex94pH and xcex94xcexa8. In studies carried out in support of the present invention, attempts to identify additional members of the vesicular monoamine and ACh transporter family, including low stringency hybridization, polymerase chain reaction (PCR) amplification of the conserved domains using degenerate oligonucleotide primers, and search of the available databases have not yielded additional members capable of transporting amino acid transport, suggesting that the proteins which effect vesicular amino acid transport may be structurally quite different from those that transport the classical transmitters.
Amino acid neurotransmitters are now recognized to play important roles in neurotransmission and neuromodulation in the central and peripheral nervous systems of vertebrate, as well as in invertebrate, species. For example, enhancement of gamma-aminobutyric acid (GABA) transmission is associated with a number of activities, including, but not limited to anticonvulsant and sedative-hypnotic activities in the mammalian and muscle relaxation. Likewise, glycine is an inhibitory neurotransmitter, and enhancement of glycine transmission may result in anticonvulsive activity in the central nervous system. In contrast, the excitatory amino acid neurotransmitters glutamate and aspartate are pro-convulsive; consequently, interference with glutamate or aspartate-mediated transmission may provide anti-convulsant effects and/ori CNS depression (McElver, et al., 1996).
Drugs which either enhance or inhibit uptake of the classical neurotransmitters into vesicles are known to provide useful therapeutic effects. It would be useful to identify similar drugs which affect amino acid transmitters via vesicular regulatory mechanisms. The present invention identifies a family of neuronal amino acid neurotransmitter transporters that effect loading of synaptic vesicles. According to an important aspect of the present invention, these transporters are sufficiently dissimilar from known monoamine or cholinergic transport proteins to define a new family of transporter proteins. These and other aspects of the invention are described herein.
According to one aspect, the invention is directed to a novel family of amino acid synaptic vesicle transporter proteins, exemplified by proteins having amino acid sequences which have substantial sequence identity to a protein having the sequence designated herein as UNC-47 and provided as SEQ ID NO: 1.
According to a related feature, the invention includes an isolated amino acid synaptic vesicle transporter protein which contains at least 10 putative transmembrane domain regions, each region having substantial sequence identity to at least one of transmembrane domain regions selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. According to an important feature of the invention, these transmembrane domains are highly conserved in members of the synaptic vesicle amino acid transporter protein described by the present invention. In a preferred embodiment, the amino acid sequence of the transporter protein of the invention is substantially identical to the transporter protein amino acid sequence designated herein as RUNC-47 and provided as SEQ ID NO: 2.
In one aspect, the invention provides an isolated nucleic acid encoding an amino acid synaptic vesicle transporter protein as exemplified by the coding regions of UNC-47 and RUNC-47 (SEQ ID NO:3 and SEQ ID NO:4, respectively), or is complementary to such an encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
According to a related embodiment, the invention includes isolated DNA fragments that encode the synaptic vesicle amino acid transporter proteins described above. According to an important feature, such fragments include a 3xe2x80x2 untranslated region, having substantial sequence identity to the 3xe2x80x2 untranslated region presented in SEQ ID NO: 4.
The invention also includes recombinant expression vectors that encompass the DNA fragments just described, as well as recombinant cells transfected with such vectors. The invention further includes antibodies directed to the proteins described above.
In yet another related aspect, the present invention includes methods of identifying a candidate compound capable of modulating amino acid transport into synaptic vesicles in the nervous system. According to this aspect of the invention, the method includes the steps of: (a) contacting a test compound with a transporter protein with an amino acid sequence which is substantially identical to SEQ ID NO: 2, under conditions in which the activity of the transporter protein can be measured; (b) measuring the effect of the test compound on the activity of such transporter protein; and (c) selecting the test compound as a candidate compound if its effect on the activity of the transporter protein is above a selected threshold level.
According to a preferred embodiment, this method is carried out in the membrane of a recombinant eukaryotic cell (i) transfected with a recombinant expression vector, comprising e.g., a DNA fragment encoding an amino acid synaptic vesicle transporter protein with an amino acid sequence which is substantially identical to SEQ ID NO: 2 or fragments thereof, which is operably linked to a regulatory sequence capable of promoting expression of the protein in a selected host, and (ii) capable of expressing the protein and processing the transporter protein for incorporation into the cell membrane. Generally, synaptic vesicle uptake of the selected neurotransmitter is measured in such an assay.
In still another related aspect the invention is directed to treatment methods, for example, treatment for a neuropsychiatric condition characterized by neuronal deficiency of GABA. According to this aspect, a subject is given a pharmaceutically effective amount of a compound capable of enhancing amino acid transport into synaptic vesicles via a transporter protein characterized by substantial sequence identity to SEQ ID NO: 2.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.